Directional acceleration vector-driven displacement of fluids (DAVD-DOF)

ABSTRACT

Centrifugal analyzer and method for staining biological or non-biological samples in microgravity, wherein the method utilizes an increase in weight of a fluid sample as a function of g-load, to overcome cohesive and frictional forces from preventing its movement in a preselected direction. Apparatus is characterized by plural specimen reservoirs and channels in a slide, each channel being of differing cross-section, wherein respective samples are selectively dispensed, from the reservoirs in response to an imposed g-factor, precedent to sample staining. Within the method, one thus employs microscope slides which define channels, each being of a differing cross-section dimension relative to others. In combination therewith, centrifugal slide mounting apparatus controllably imposes g-vectors of differing magnitudes within a defined structure of the centrifuge such as a chip array.

RELATED APPLICATIONS

[0001] U.S. Pat. No. 6,008,009

[0002] CENTRIFUGE-OPERATED SPECIMEN STAINING METHOD AND APPARATUS,

[0003] Dated Dec. 28, 1999, Inventors Mark S. F. Clarke and Daniel L.Feeback.

[0004] U.S. DISCLOSURE DOCUMENT NO. 470956,

[0005] RECORDED U.S. PATENT AND TRADEMARK OFFICE MAR. 16, 2000, AuthorsMark S. F. Clarke and Daniel L. Feeback.

U.S. GOVERNMENT RIGHTS

[0006] This invention was made with U.S. Government support undercontract NCC9-41 awarded by NASA. The Government has certain rights inthis invention.

BACKGROUND OF INVENTION

[0007] In the past, the inventors herein have patented a device for thestaining of biological samples in microgravity aboard a spacecraft,known as the centrifuge operated slide stainer. This device andtechnology was developed in response to the need for real-time analysisof crew member blood samples and microbiological monitoring of samplesobtained from the environmental systems of a spacecraft. That method andassociated apparatus, hereinafter COSS, appear in U.S. Pat. No.6,008,009, dated Dec. 28, 1999, entitled Centrifuge-Operated SpecimenStaining Method and Apparatus, inventors Mark S. F. Clarke and Daniel L.Feeback. Further study of the need for analysis of biological samplesduring space flight suggested that a greater range of sample types andstaining protocols would have to be accommodated by any stainingtechnology selected for flight. This was due not only for medicaloperations reasons and environmental monitoring, but also for theanticipated increased requirement for analysis of biological samplesobtained from science experiments conducted aboard the establishedInternational Space Station. In order to accommodate such an increase indemand, the possibility of miniaturizing the COSS technology wasinvestigated so that a greater range of samples and staining protocolscould be accommodated with no, or less, impact on crew time, solid wasteproduction and upmass than that of the aforesaid technology. The needfor real-time analysis of biological samples during space flight isexemplified by the requirement for a differential white cell count DWCC,a critical medical diagnostic tool which can be used to distinguishbetween various conditions that induce alterations in the total numberand type of white blood cells produced by the human body. For example, aDWCC can be used to distinguish between bacterial or viral infections,in the differential diagnosis of an allergic reaction or to detect thepresence of myeloproliferative disorders or leukemia. Microgravityexposure during space flight results in hemodynamic changes in crewmembers, which in turn impacts the production of white blood cells.Heretofore, no data are available to establish the “normal baseline” forwhite blood cell production in microgravity. Without first knowing theextent to which microgravity exposure impacts white blood cellproduction, or secondly the proper “microgravity baseline” for a normalhealthy crew member in space, it is quite possible that a bacterial orviral infection may be overlooked or misdiagnosed, or that a potentiallymuch more serious problem, such as leukemia, may be attributed to abacterial or viral infection in a particular crew member. In addition,the requirement for microbiological screening of both medical andenvironmental samples during space flight, which can only beaccomplished by utilizing specific staining techniques for microbeidentification, is a second example of the need for real-time analysiscapabilities using standard staining techniques on-orbit. At present, itis impossible to perform a DWCC while aboard a space craft. Whole bloodsmears have been produced in microgravity, but as yet it has remainedimpossible to perform a DWCC without returning the blood smear to Earth.Due to the limited life span of such smears, it is impossible to make adefinitive statement with regard to the effect of microgravity exposureupon white blood cell profiles based on such samples. Until real-timeperformance and analysis of a DWCC can be achieved aboard the spacecraft, critical crew health information thus remains unobtainable.

[0008] In a terrestrial setting, a differential white cell count isobtained by preparing a blood smear on a glass slide, fixing the cellsin the smear to the surface of the slide, staining the cells with ahistochemical stain followed by washing the slide in a clean buffersolution prior to viewing under the microscope where a “differential”white blood cell count is made by morphological criteria. The protocoloutlined above is a simple and universally used technique to perform aDWCC. However, this technique requires the use of liquid buffersolutions, including fixatives and dye solutions. While this techniqueis performed easily on Earth, the problems associated with liquidhandling in microgravity make such a task nearly impossible. Pastattempts at solving this problem have included several “cell stainers”which were tested by NASA or its contractor personnel but have sinceproved unsuitable for use in microgravity. The first attempt was a slidestainer which flew aboard Sky Lab. This device proved very cumbersome,required large volumes of buffer solutions and had limited use due toprecipitate formation in the staining solutions which blocked theintricate tubing arrangement required to apply the staining solutions tothe blood smear. A second attempt was based upon an airtight chamberdesign which contained a blood smear slide, into which buffer solutionsand/or staining solutions were introduced using a vacuum system. Systemoperation relied upon a series of one-way and two-way valves in order toachieve an efficient vacuum into which the staining solutions wereintroduced by hypodermic syringe. The original technology used ahand-held squeeze bulb to create the vacuum which proved inadequate. Alater version incorporated mechanical pumps to provide both vacuumproduction and syringe emptying. The hand-operated version of thistechnology, although shown to work on the ground and which passedinitial testing aboard the KC-135 parabolic aircraft, did not fulfillits potential and has since been shelved as a viable solution to slidestaining on-orbit, not least because of its requirement for substantialcrew interaction and crew time.

THE PRIOR ART

[0009] INVENTOR PAT. NO. DATE TITLE R. Hughes et al. 3,352,280 1967Centrifugal Apparatus For Slide Staining van Duijn 4,192,250 1980Valve-Centrifuge Peterson et al. 4,225,558 1980 Fluid sample testapparatus and fluid sample cell for use therein Eberle 4,612,873 1986Centrifuge chamber for cytodiagnostic investigation of epithelial cellscontained in a sample Molina et al. Article* 1990 Applied MicrobiologyGram Staining Apparatus Kopf-Sill 5,160,702 1992 Analyzer with improvedrotor structure Nilsson et al. 5,286,454 1994 Cuvette Hayes 5,589,4001996 Method of distributing material onto a microscope slide of a largecytology sample chamber Kelley et al. 5,679,154 1997 Cytology CentrifugeApparatus Clarke et al. 6,008,009 1999 Centrifuge-operated specimenstaining method and apparatus

SUMMARY OF INVENTION

[0010] In the present technology, termed Directional AccelerationVector-Driven Displacement of Fluids DAVD-DOF the same end point, namelysequential filling and emptying of a staining chamber, is achieved usinga network of reservoirs and connecting tubes created on a single slide.However, unlike the earlier COSS technology, fluid displacement isachieved by utilizing the weight of the fluid itself, rather than aweighted plunger, to force the fluid through a network of channelsbetween fluid reservoirs. Selective emptying of separate fluidreservoirs is achieved by altering the cross-sectional area of thechannel which connects the reservoirs. As cross-sectional area of thechannel decreases, the g-force required to bring about fluiddisplacement through the channel is increased. This approach reduces theoverall size of the equipment required to perform a staining protocol inmicrogravity as well as reducing the amount of staining reagent requiredfrom approximately 3 milliliters per reagent in the original COSStechnology to less than 20 microliters in the DAVD-DOF technology. Asthe staining protocol is carried out in a centrifuge at g-levels above1× g, the problems associated with liquid handling in microgravity, suchas air/liquid mixing and bubble formation do not occur. This is due tothe fact that a liquid is much heavier than air in the increasedacceleration field produced by its rotation in a centrifuge, therebyproducing a clear and defined liquid/air interface, an attribute commonto both the original COSS and present DAVD-DOF technologies. Thetechnology described herein is thus based upon the concept that fluids,in this case, staining reagents used for biological sample analysis, canbe transferred from one reservoir to another through a connectingtube/channel by applying a gravity vector or acceleration vector in thedirection of the required movement. This concept is essentiallydifferent from the original COSS device, U.S. Pat. No. 6,008,009. Thatsystem utilizes a weighted plunger designed to force fluid from onecontainer to another at a constant level of hypergravity maintained in astandard swing-bucket centrifuge. This arrangement allows the sequentialfilling and emptying of a staining chamber containing a microscope slideon which a biological sample is mounted. In this centrifugal analysis,the principle of invention is stated as optimizing defined dimensionalchannels of a specimen slide to effect controlled movement of specimenfluids therein by defined g-forces.

DESCRIPTION OF PREFERRED EMBODIMENTS

[0011]FIG. 1 depicts schematically the G-Force required to displace 50microliters of fluid down equilength channels of differentcross-sectional areas. These values were determined through manualexperimentation in which fluid was displaced between two reservoirsconnected via a capillary tube (i.e. a hypodermic needles of knowncross-sectional area) using a centrifuge to apply hypergravity g-vectorsof increasing intensity, in a direction aligned with the length of thecapillary tube, to cause fluid displacement. The results of theseexperiments validate that there exists an inverse relationship betweenthe cross-sectional area of a capillary tube and the g-force which mustbe applied to cause fluid displacement through the capillary tube. Thissimple effect, utilizing an increase in weight of a fluid as a functionof g-load to overcome the cohesive and frictional forces preventing itsmovement in a particular direction is the basis of the DAVD-DOFtechnology, See FIG. 1.

[0012] In the experiments, the results of which are summarized in FIG.1, a reservoir containing 50 microliters of fluid was connected to asecond empty reservoir via a hypodermic needle of known cross-sectionalarea and placed in a centrifuge, with the full reservoir disposedclosest to the central spindle of the centrifuge and the capillary tubealigned perpendicularly to the central spindle (i.e., the direction ofmaximum g-load generated by centrifugal rotation). At 1× g, the surfacetension (i.e. cohesive forces of the liquid itself and the frictionalforces between the liquid and walls of the reservoir and the needle)prevented the liquid from passing through the needle as a consequence ofits own weight. As the RPM of the centrifuge, and hence g-force placedon the liquid was increased, however, the weight of the liquid alsoincreased. When the weight of the liquid was great enough to overcomethe forces preventing it from passing down through the needle in thedirection of the acceleration vector (i.e. the surface tension of theliquid and the frictional forces between the fluid and the sides of thetube), the liquid was displaced from the full reservoir, through theneedle, to the second reservoir. By varying both the internal diameterof the needle and the revolutions per minute (RPM) of the centrifuge andhence g-force on the constant volume of liquid, in this case 50microliters, the inverse relationship between the cross-sectional areaof a capillary tube and the g-force was established. See FIG. 1.

[0013] To test the concept that the DAVD-DOF principle works at thescale envisioned for creating an array of fluid reservoirs andconnecting fluid channels on a microscope slide, microchannels andreservoirs were constructed on a commercially available microscope slidecoated with a 10 micron thick Teflon® mask, FIGS. 4A and 4B. The Teflon®mask initially formed a series of wells 28 on the surface of the glassslide, FIG. 4A. A 60 micron wide channel 29 was created between the twowells by removing the Teflon® masking material with a razor blade FIG.4B. An access channel 30 of approximately 1 mm width and a vent channel31, sixty microns wide, was created in order to load Reservoir 32 withfluid. A similar vent channel 33, sixty microns wide, allows airdisplacement from Reservoir 34 when fluid is displaced by the DAVD-DOFprinciple from Reservoir 32. A glass cover-slip was then glued to thesurface of the Teflon® mask, to create a device, a typical cross-sectionof which is presented in FIG. 2D. Using the large access channel 30,reservoir 32 was completely filled with a colored liquid without fluidentering reservoir 34 through the 60 micron thick connecting channel 29.When this slide was then centrifuged at a g-level of approximately 22×g, the colored liquid was displaced from reservoir 32 into reservoir 34via the connecting 60 micron channel 29.

[0014] In the embodiment represented by FIGS. 4A and 4B, channel 29connecting reservoir 32 to reservoir 34 is approximately 60 micronsacross and 10 microns deep. The micron sized dimensions of channel 29are designed to negate the effects of capillary action. At this scale(i.e. the micro-fluidic scale, in which the cross-sectional area of thechannel is less than 1000 square microns), the surface tension of thefluid in reservoir 32 at the entrance to channel 29 is the over-ridingforce, rather than the capillary action forces generated by channel 29.Hence, fluid does not enter channel 29 until additional forces areapplied to overcome this surface tension. At higher g-values, however,the weight of the fluid applied in a particular direction overcomes thesurface tension effect at the entrance of the channel 29, allowing fluidto enter channel 29 and to flow to reservoir 34. Access channel 30, bycontrast, is approximately 1 mm in width and 10 microns deep. Suchlarger dimensions facilitate the filling of reservoir 32, in part, bycapillary action.

[0015]FIGS. 2A and 2E are diagrammatic representations of an embodimenta staining apparatus created on a glass microscope slide utilizing theDAVD-DOF principle. A series of fluid reservoirs connected by channelsof different cross-sectional dimensions are created on the surface of aglass microscope slide, FIG. 2A. This pattern of inter-connectedreservoirs and corresponding channels can be created on the slide byeither etching of the surface of the glass slide itself and bonding alayer of glass to the etched surface of the slide, FIG. 2B, cutting amask out of a uniformed thickness sheet of inert material, such asTeflon®, which is then sandwiched and bonded between two layers ofglass, FIG. 2C, or etching away of an inert material, such as Teflon® orpolycarbonate, previously applied and bonded to the surface of the glassslide as a uniform thickness layer followed by bonding a glass layer tothe etched surface, FIG. 2D. Alternatively, the same arrangement can beaccomplished by either excimer laser-cutting or photolithography of apredetermined pattern of reservoirs and connecting tubes to create asheet of inert material of uniform thickness which is then sandwichedand bonded between two layers of glass, FIG. 2C. In FIG. 2E, glasslayers 62 and 64 have been bonded to the etched or lithographedsurface(s) of the slide using, for example, UV-activated adhesive orelectrostatic bonding, thereby creating a sealed array of fluidreservoirs and connecting tubes of defined dimensions with an open-facedstaining reservoir 8. Fluid is then dispensed into the fluid reservoirsusing a micro-needle and displacement volumetric pipette via theirrespective access ports 6, displaced air exiting through theirrespective air vents 7. The sample, e.g. isolated cells, blood smear ortissue section, is first attached onto one surface of a standard 22 mmsquare glass cover-slip 66 which is then placed sample side down overthe staining chamber. The cover-slip is then attached to the surface ofthe glass slide with air-activated adhesive, such as methyl acrylate,pre-applied to the surface of the slide surrounding the stainingchamber. The adhesive is protected from hardening by a gas-impermeablemembrane which is removed immediately prior to placement of thecover-slip. This arrangement is the final step in producing a sealedinter-connected array of fluid reservoirs and connecting tubes ofdifferent cross-sectional dimensions on the slide.

[0016] The slide is then placed on a centrifuge consisting of a flatspinning disc as in plan FIG. 3A. Sequential fluid displacement from thereservoirs, in the depicted configuration all reservoirs contain anequal volume of liquid, into the staining chamber is achieved byincrementally increasing the RPM/g-force produced by the spinning disccentrifuge or SDC.

[0017] Where the text and/or figures indicate equal volume of reservoirsand/or equal length of channels, alterations in volumes and/or lengthsmay be incorporated without derivation from the spirit of thisinvention.

[0018] Principle of Operation:

[0019] In FIG. 2A, each fluid reservoir 1, 2, 3, 4 and 5 of slide 15 hasan access port 6 through which a known volume of fluid can be dispensedinto the reservoir using a micro-volumetric displacement pipette. Asdiscussed previously in relation to FIGS. 4A and 4B , the dimensions ofaccess ports 6 are sufficiently large (e.g., approximately 1 mm wide by10 microns deep) to facilitate the filling of reservoirs 1, 2, 3, 4 and5, in part, by capillary action. Air displaced from the reservoirsduring fluid filling exits via the air vent channels 7. By way ofexample, a biological sample such as a blood smear or a cell culture isattached to a microscope slide cover-slip, FIG. 2E, and the cover-slipis placed sample-side down, over the top of the staining chamber 8 whichhas been left uncovered. The cover-slip, with mounted sample downwards,is then bonded to the uncovered area of the etched surface of the slide,as previously described, thereby creating a completely sealed array ofreservoirs and connecting channels. When g-force is applied to the slide15 in the direction indicated in FIG. 2A, fluid tends to move in thedirection of the g-vector. As a consequence, fluid will be displacedfrom the reservoirs 1-5 when the weight of the fluid in each reservoirovercomes the surface tension of the fluid itself and the frictionforces between the fluid and the walls of both the reservoir andconnecting channels. As G-force increases, sequential emptying ofreservoirs 1 through 5 into the staining chamber 8 will occur. Forexample, if reservoirs 1, 2, 3, 4, and 5 are filled with equal volumesof fluid, reservoir 1 will empty into the staining chamber at the lowestg-force of any of the reservoirs 1-5 as that connecting channel 9,between the reservoir 1 and the staining chamber 8, has the largestcross-sectional area of the channels 9 through 13. As discussedpreviously in relation to FIGS. 4A and 4B, channels 9, 10, 11, 12, and13 have cross-sectional area's small enough (i.e., less than 1000 squaremicrons) that liquid movement by capillary action along these channelsis prevented by the surface tension of the fluid itself. It is only whena gravitational vector of a high enough g value is orientated along thechannel that the surface tension forces at the entrance of the channelare overcome and the liquid is forced through the channel into thereaction chamber.

[0020] This principal prevails hereinafter. As fluid displacement occursunder hypergravity conditions, liquid/air mixing is precluded duringfluid displacement, due to the large differential between the weight ofthe fluid and the weight of the air under such hypergravity conditionswhich ensures a highly discrete air-liquid interface and prevents theformation of gas bubbles that may otherwise produce air locks in thechannels and chamber/reservoirs. Furthermore, each reservoir is equippedwith vents that allow air to escape as fluid enters the reservoir. Thesevents prevent the formation of bubbles and air locks in channels due toincreased air pressure in the respective reservoirs. In FIG. 2a, forexample, staining chamber 8 is vented by vents 7′ and the waste reagentreservoir 17 is vented by vents 7″. When the g-force required to emptythe reservoir 1 is achieved, the centrifuge is slowed to a minimalrevolution rate for a pre-determined time in order for the fluid to bein contact with the sample in the staining chamber 8. After thispre-determined period of time, micro-valve 14, supplied with electriccurrent via electric contacts 18 on the side of the slide 15, is openedin channel 16 and the centrifuge is spun up again to a g-force belowthat required for emptying of reservoir 1. The large cross-sectionalarea of exhaust channel 16, at least 5 times that of channel 9, ensuresa rapid emptying of the staining chamber 8 into the waste reagentreservoir 17 at a relatively low g-force compared to that required foremptying of the fluid reservoirs 1-5. The micro-valve 14 is then closedand g-force is increased until emptying of reservoir 2 is achieved, atwhich time the g-force is again lowered to a nominal value to allow thefluid to react with the sample in staining chamber 8. After apre-determined period of time the g-force is again increased; themicro-valve 14 is again opened to allow emptying of staining chamber 8.This cycle is repeated until all five reservoirs have been sequentiallyemptied into the staining chamber 8 and then collected in the wastereagent reservoir 17, leaving an appropriately treated sample in thestaining chamber. As both sides of the staining chamber are made fromoptical quality glass the sample, contained in its staining chamber canbe directly viewed under the microscope as would be a normal microscopeslide.

[0021]FIG. 3A and 3B are diagrammatic representations of a firstembodiment of the spinning disc centrifuge for use with the DAVD-DOFslide depicted in the schematics of FIGS. 2A-E.

[0022] Controllable g-force in a single plane is achieved by using arotating disc on which the slide 23 is positioned so that the fluidreservoirs on the slide are disposed closest to the central spindle 21of the centrifuge FIG. 3A. A microprocessor 19 and a power supply 20 formicro-valve operation are housed within the spinning disc nearest thecentral spindle 21 to reduce any effects of hypergravity upon theelectronic components. Power is supplied to microprocessor 19 from powersupply 20 via solid state electrodes 26. Power, required for opening andclosing of the micro-valve on the slide is supplied to the micro-valvevia solid state electrodes 22 emanating from the microprocessor 19.Slides 23 are immobilized in the centrifuge by recesses 24 in thesurface of the spinning disc and a restraining disc 25, FIG. 3B, issecured over the central spindle 21, to ensure that the slides are notdisplaced during centrifugation and that the direction of the g-vectoris constant with regard to orientation of the slide. Centrifuge RPM, andhence g-force, is controlled by the microprocessor unit 19 that controlsa variable speed electric motor, allowing accurate modulation of g-forceby controlling motor RPM. In this fashion, the micro-processor unitcommunicates with the micro-valve on the slide and the centrifuge motorin order to control the required changes in g-force at the appropriatetimes during the staining protocol i.e. low rpm when the micro-valve isopened in order to empty the staining chamber; incremental increases ing-force for sequential fluid reservoir emptying when the micro-valve isclosed. Although any micro-valve device could be used, one viablemicro-valve that has been found suitable operates on the principle of amicro-force array which expands into the connecting channel when anelectric current is applied to the array, thereby blocking the channel.

[0023]FIGS. 5A and 5B are diagrammatic representations of a secondembodiment of the spinning disc centrifuge for use with a silicon wafermicro-array which utilizes the present DAVD-DOF principle. FIGS. 5A and5B are described below.

[0024] A DAVD-DOF microarray 35 is secured in place by clamps 35 aattached to a ¾ volume spheroid 36. This spheroid is free to rotate 180degrees in the vertical plane about a supporting axle 37 attached to arotating cog-ring 38. Cog ring 38 is free to rotate 360 degrees in thehorizontal plane. This composite structure is supported in a spinningdisc centrifuge 47 which rotates about central spindle 45 drive shaft 51assembly, at a predetermined rate to produce a g-vector of a definedlevel in the direction indicated by the arrow 46. By altering theorientation of the microarray 35 relative to the direction of theg-vector 46, fluid movement between reservoirs can be achieved in threedimensions by aligning the channel connecting said reservoirs to thedirection of the g-vector 46. As shown in FIG. 5A and supporting cutawayFIG. 5C, movement of the spheroid 36 in the vertical plane is achievedby activation of a screw drive 39 engaged to an integral cog 48 locatedon the supporting axle 37. The screw drive is powered by a power supply40 located on the surface of the rotating cog ring 38 and orientation inthe vertical plane is controlled by a programmable microprocessor 41also located on the surface of the same rotating cog ring 38. Byengaging the screw drive with the integral cog 48 on the supporting axle37 for a predetermined period of time, the orientation of the microarrayin the vertical plane can be accurately controlled in one degreeincrements. The orientation of the microarray 35 relative to theg-vector 46 in the horizontal plane is controlled by a second drive cog49 and a freely rotating support cog 50 sandwiched between upper andlower layers of the spinning disc centrifuge 47. The drive cog 49 ispowered by a second screw drive 42 also located on the upper surface ofthe spinning disc centrifuge 47. Rotation of the rotating cog ring 38through 360 degrees in one degree increments is achieved by engaging thescrew drive 42 with the drive cog 49 for a predetermined period of time.See FIG. 5B. The drive cog 49, in turn, is engaged to the rotatingcog-ring 38 in which the ¾ spheroid 36, upon which the microarray 35rests, is supported. The screw drive 42 is activated by a power supply43 and controlled by a programmable microprocessor 44 located on thesurface of the spinning disc centrifuge 47. By using a predeterminedcombination of independent rotational movements of both the rotatingcog-ring 38 and the ¾ spheroid 36, the microarray 35 can be placed inany orientation relative to the g-vector 46 in three dimensions. Thisability allows connecting channels between fluid reservoirs created onthe surface of the microarray 35 to be aligned with the g-vector 46 andhence bring about fluid movement in this direction along the channel.The ability to rate the microarray in the vertical plane allows amicroarray consisting of multiple layers, see FIG. 6, connected bychannels 53 between the layers, to be employed. FIG. 6 presents a crosssection of a DAD-DOF microarray, such as those presented in FIGS. 2B,2C, and 2D, in which a 3-dimensional microarray has been created bysandwiching a 3-dimensional inert layer 54, created usingphotolithography techniques, between two glass layers 55 using andadhesive bond 56. Fluid movement between layers is achieved by rotatingthe microarray so that the channel connecting the separate layers isaligned to the direction of the g-vector. This has the advantage ofallowing the complete volume of the microarray for purposes of sampleprocessing and/or reagent storage rather than some portion on a singlesurface of the microarray.

[0025] As previously discussed in relation to FIGS. 3A and 3B, thespinning disc centrifuge can be equipped with a microprocessor toautomate control of centrifuge RPMs and to automate actuation ofmicro-valves upon each of the respective microarray devices. In theembodiment presented in FIG. 5A, the programmable microprocessor 44 canbe used to control the 360 degree rotational positioning of the cog-ringand the 360 degree positioning of the ¾ spheroid, in addition tocontrolling centrifuge RPMs and micro-valve actuation. Communicationbetween microprocessor 44 and the cog ring screw drive 42 could beaccomplished by solid state electrodes 60 mounted upon the spinning disccentrifuge 47. Transmitter/receiver 62 communicates with miniaturetransmitter/receiver 64, mounted upon the cog-ring, and miniaturetransmitter/receiver 66, mounted upon the ¾ spheroid, to controlpositioning of the ¾ spheroid, and actuation of microarray micro-valves,respectively. Transmitter/receiver 62 receives power from power supply63 via solid state electrodes 65 and is controllable connected tomicroprocessor 44 by solid state electrodes 61 mounted upon the spinningdisc centrifuge 47. Although not indicated in FIG. 5A to avoidcongestion within the figure, transmitter/receiver 64 receives powerfrom power supply 40 and is controllably connected to microprocessor 41by solid state electrodes mounted upon the cog-ring 38. Microprocessor41 controls actuation of screw drive 39 by solid state electrodesmountedupon the cog-ring 38, as well. Furthermore, transmitter/receiver 66receives power from power supply 68 and controls actuation of microarraymicro-valves by solid state electrodes mounted upon the ¾ spheroid 36.

[0026] The DAVD-DOF technology described here is a preferred means ofdisplacing fluids from one location to another without the production ofair bubbles in the solution. As such, this approach overcomes one of thecentral problems associated with any liquid handling in microgravity,namely air/liquid mixing. In addition, this approach allows thedisplacement of fluid volumes which at 1× g would form only as a liquiddroplet due to surface tension effects. The direct advantage of thistechnology is that it provides a means of biological sample processing,utilizing staining techniques fully validated in terrestriallaboratories, as the staining protocols and liquid reagents utilizedwith the DAVD-DOF slide apparatus are identical to those used on Earthin reference laboratories. The DAVD-DOF slide technology is small,lightweight, versatile, i.e. any staining protocol, standard orotherwise, may be accommodated by this technology, uses small reagentvolumes, produces no solid or liquid waste apart from the slide itself,requires little crew-time for operation and is modular in design. Thesecharacteristics, plus the totally automated function of the device oncethe slide has been placed in the centrifuge, make the DAVD-DOF slidetechnology very attractive for use aboard ISS for crew health andenvironmental systems monitoring as well as for scientific researchamong various scientific disciplines.

The inventors hereby claim: 1 In real-time centrifugal analysis for thestaining of fluid samples in microgravity, a method of directionalacceleration, vector driven displacement of the samples for sequentialfilling and emptying of a staining chamber: as applied to at least oneslide, sequentially filling and emptying fluid samples in a slidestaining chamber by incrementally increasing an applied g-force througha network of supply reservoirs and discrete connecting channels formedon said one slide, wherein displacement of the fluid samples dependsupon volume and weight of the fluid therein, and as the preselectedcross-sectional area of a given discrete channel is modified relative toother channels, the g-force required for sample fluid displacementtherethrough is controllably activated, whilst simultaneously air isexhausted from the staining chamber, thereby reducing the size of theequipment required as well as quantity of staining reagent, thusproducing a clear and defined liquid/air interface. 2 The method ofclaim 1 including an initial step of emptying at least one separatefluid-filled supply reservoir of the slide by utilizing the volume andweight of fluid in a given reservoir at a particular g-level to overcomesurface tension of the fluid and frictional forces between the fluid andwalls of the connecting channels. 3 The method of claim 2 wherein anadded step of collecting waste reagent from the staining chamber in onewaste reagent reservoir of the slide comprises applying a g-force belowthat required for emptying other reagent reservoirs. 4 The method ofclaim 2 wherein an added step of sequentially emptying a series of fluidfilled supply reservoirs into a separate supply reservoir, comprisesincreasing volume and weight of fluid sample contained in a givenreservoir, whilst maintaining constant the cross-sectional area of theconnecting channel, as well as maintaining constant the applied g-force.5 The method of claim 4, wherein the added step of collecting wastereagent from the staining chamber in one waste reagent reservoir of theslide comprises applying a g-force below that required for emptyingother connected reagent reservoirs. 6 The method of claim 1 wherein anadded step of sequentially emptying of a given supply reservoir into aseparate supply reservoir comprises maintaining the volume and weight ofthe liquid constant, whilst decreasing the cross-sectional area of itsconnecting channel and simultaneously increasing the applied g-force. 7The method of claim 6 including an added step of collecting wastereagent from the staining chamber simultaneously passing same to a wastereagent reservoir of the slide by applying a g-force below that requiredfor emptying other connected reagent reservoirs. 8 The method of claim3, 5, or 7 including the added step of controlling the flow of wastereagent to a waste reagent reservoir by means of a micro-valve toselectively block the channel connecting the staining chamber and thewaste reservoir. 9 The method of claim 1 including transferring bychannel connection a given fluid sample from one supply reservoir toanother, precedent to transfer of said fluid sample to a stainingchamber, by applying a controlled gravity vector in the transferdirection of required movement, whilst avoiding creation of air bubblesin sample solution. 10 The method of either claim 1, 2, 3, 4, 5, 6, 7 or9 wherein the fluid samples are of biological composition. 11 The methodof either claim 1, 2, 3, 4, 5, 6, 7 or 9 wherein the fluid samples areof non-biological composition. 12 Centrifugal analyzer for real-timecentrifugal analysis of biological or non-biological fluid samples inmicrogravity, wherein directional acceleration of the samples is imposedby vector driven displacement of said samples, comprising: a) a specimenmicroscope slide, a network of specimen slide supply reservoirs thereinand at least one common staining chamber thereon with discreteconnecting channels, each channel defining a conduit of variantcross-section from channel to channel, said conduits interconnectingrespective supply reservoirs with said staining chamber; b) a source ofvariable centrifugal force applied to respective slides wherein as thecross-sectional area of one channel decreases, or increases, relative toanother, the g-force required for sample fluid displacement in a givenchannel may be altered from increased to decreased, and vice versa. 13The centrifugal analyzer of claim 12 including a control means toincrease and/or decrease the g-forces required for sample displacementthrough connecting channels. 14 The centrifugal analyzer of claim 12including a programmed control means to increase and/or decrease theg-forces required for sample displacement through connecting channels.15 The centrifugal analyzer of claim 12 including a manually operatedcontrol means to increase and/or decrease the g-forces required forsample displacement through connecting channels. 16 The centrifugalanalyzer of claim 12 including a waste reagent reservoir connected to anoutlet of said staining chamber, the cross section of its connectingchannel being greater than that of other respective channels connectingsupply reservoirs to the staining chamber. 17 The centrifugal analyzerof claim 12, 13, 14, or 15, including a waste reagent reservoir definedby the slide in exhaust vector connection with the staining chamber,whereby as the cross section of a given channel is altered in dimensionrelative to the cross sections of others, the control means isaccordingly altered to effect g-forces required for sample displacement.18 Apparatus according to claim 12, 13, 14, or 15 whereby a givenchannel of the slide defines a conduit of uniform cross section which isaltered in area relative to that of others and said control means isresponsive accordingly, to modify the displacement by selectivelyaltering g-forces required for fluid sample displacement. 19 Thecentrifugal analyzer of claim 12 wherein plural supply reservoirs areinterconnected by a microchannel capillary tube, each said reservoirincluding a separate air vent for air displacement therefrom. 20Apparatus according to claim 16 or 17 wherein a micro-valve within theoutlet channel of said staining chamber is used to control flow betweensaid staining chamber and said waste reagent reservoir. 21 A spinningdisc centrifuge, for use in real-time centrifugal analysis for thestaining of fluid samples in micro-gravity, comprising: a) a rotatingdisc; b) a first means within said disc to receive and restrainmicro-array slides in a fixed position; c) a second means within saiddisc to orient said receiving and restraining means relative to thedirection of gravitational force caused by rotation; said first andsecond means having independent connection to each other. 22 A spinningdisc centrifuge, as recited in claim 21, wherein said means to orientsaid receiving and restraining means is a cog ring, mounted within saiddisc, capable of rotating plus or minus 360 degrees within the sameplane as said rotating disc. 23 A spinning disc centrifuge, as recitedin claim 22, wherein said means to receive and restrain micro-arrayslides is a ¾ spheroid, capable of rotating plus or minus 360 degreesabout a supporting axle attached centrally within said cog ring. 24 Aspinning disc centrifuge, as recited in claim 22, further comprising: a)a drive cog, disposed between the upper and lower surfaces of saidrotating disc centrifuge, with gear teeth that engage gear teeth on theouter perimeter of said cog ring; b) a cog ring drive motor, mountedupon surface of said rotating disc, that engages said drive cog; wherebyas said cog ring drive motor is engaged, said drive cog is rotated andincremental rotation of said cog ring results. 25 A spinning disccentrifuge, as recited in claim 22, wherein the cog ring is rotated by amotorized drive, power supply, and control means mounted to saidrotating disc. 26 A spinning disc centrifuge, as recited in claim 23,further comprising: a) an integral cog located on the supporting axle ofsaid ¾ spheroid; b) a spheroid drive motor, mounted upon surface of saidcog ring, that engages said integral cog, whereby as said spheroid drivemotor is engaged, said integral cog is rotated and incremental rotationof said ¾ spheroid results. 27 A spinning disc centrifuge, as recited inclaim 23, wherein said ¾ spheroid is rotated by a motorized drive, powersupply, and control means mounted to said supporting cog ring. 28 Aspinning disc centrifuge, as recited in claim 21, further comprising: a)a microprocessor controller, mounted upon said disc in operativeconnection with; b) a power supply, mounted upon said disc and supplyingpower to said microprocessor controller; c) a control means by whichsaid microprocessor controller communicates with the controller for thevariable speed motor of the centrifuge; whereby said microprocessor maycontrol the rotational speed of the centrifuge's variable speed motor,thus allowing accurate modulation of g-force by controlling motor RPM.29 A spinning disc centrifuge, as recited in claim 28, furthercomprising: a) a control means by which said microprocessor communicateswith said cog ring drive motor; whereby said microprocessor may controlthe rotational position of said cog ring. 30 A spinning disc centrifuge,as recited in claim 29, further comprising: a) a control means by whichsaid microprocessor communicates with said ¾ spheroid drive motor;whereby said microprocessor may control the rotational position of said¾ spheroid. 31 A spinning disc centrifuge, as recited in claim 28, inwhich said control means are miniature radio transmitter/receivers, onetransmitter/receiver mounted upon said rotating disc, a secondtransmitter/receiver within the controller for the centrifuge's variablespeed motor. 32 A spinning disc centrifuge, as recited in claim 29, inwhich said control means are solid state electrodes mounted upon saidrotating disc connecting said first microprocessor with said cog ringdrive motor. 33 A spinning disc centrifuge, as recited in claim 30, inwhich said control means, are miniature radio transmitter/receivers, onetransmitter/receiver mounted upon said rotating disc, a secondtransmitter/receiver mounted upon said cog ring. 34 A spinning disccentrifuge, as recited in claim 28, further comprising: 1) a controlmeans whereby the micro-valve(s) within each micro-array can be actuatedby said microprocessor. 35 A spinning disc centrifuge, as recited inclaim 34, in which said control means are solid state electrodes mountedupon said rotating disc connecting said microprocessor said micro-valveleads upon said micro-array. 36 A spinning disc centrifuge, as recitedin claim 34, in which said control means are miniature radiotransmitter/receivers, one transmitter/receiver mounted upon saidrotating disc, a second transmitter/receiver mounted adjacent to solidstate electrodes connecting said second transmitter/receiver tomicro-valve leads upon said micro-array. 37 A spinning disc centrifuge,as recited in claims 21, further comprising a means by which to precludedisplacement of the slides during centrifugation and to provide thatg-force remains constant with respect to the orientation of the slide.38 A spinning disc centrifuge, as recited in claims 21, in which themeans to preclude displacement of the slides during centrifugationcomprises a restraining disc, secured over the central spindle, thatrotates with said rotating disc.